Journal: Annals of the rheumatic diseases

Article Title: RUNX1 is expressed in a subpopulation of dermal fibroblasts and is associated with disease severity of systemic sclerosis

doi: 10.1016/j.ard.2025.10.033

Figure Lengend Snippet: TGF- β 1 increases the RUNX1 expression in SSc fibroblasts and inhibition of RUNX1 reduces ECM markers. (A) Western blot of 3 isolated fibroblasts lines treated with TGF- β 1. The blot shows all isoforms of RUNX1a, b, and c that are overexpressed under the TGF- β 1 stimulation. (B) Schematic graph illustrating the timeline for the culture and TGF- β 1 treatment of dcSSc-isolated fibroblasts, matched healthy-isolated fibroblasts, and normal human dermal (NHD) fibroblast cells. RUNX1 expression rate in samples treated with TGF- β 1 (in red) vs control (in blue) for the 24 hours after exposure. (C) Volcano plot of differentially expressed analysis of the 2 SSc-isolated fibroblast lines at 12 hours after exposure vs the baseline. (D) The pathway analysis of Reactome gene sets shows the biological pathways and processes that are significantly represented within top DEG genes of SSc-isolated fibroblast lines 12 hours after TGF- β 1 treatment vs the baseline. Data from B to D were obtained through publicly available data of GSE12493 . (E) Schematic graph showing 2 lines of SSc-isolated fibroblasts treated with siRNA against RUNX1 (siRUNX1) and nontargeting control siRNA (siNC). (F) UMAP projection and dot plot of RUNX1 and CBFB of the single-cell RNA-seq data. (G) UMAP of 10 fibroblast clusters (0–9) for siR-UNX1 and siNC. (H) Cell proportion of siRUNX1 and siNC per cluster. (I) Top 4 upregulated and downregulated marker genes per cluster. (J) Top 15 enriched pathways that are significantly represented across siRUNX1 and siNC (K) Bar plots showing the percentage of cells expressing COL1A1, FN1, COL4A1, LUM, ACTA2, LGR5, COL8A1, COMP , and THBS1 per condition (red: siRUNX1, green: siNC) or per cluster. (L) Module score for extracellular matrix organisation pathway per cluster and per condition. (M) Feature plot of the ECM module score. dcSSc, diffuse cutaneous SSc; DEG, differentially expressed gene; ECM, extracellular matrix; RUNX1, runt-related transcription factor 1; siRUNX1, siRNA targeting RUNX1; SSc, systemic sclerosis; TGF- β , transforming growth factor- β ; UMAP, uniform manifold approximation and projection.

Article Snippet: We then analysed a previously generated DNA microarray dataset (National Center for Biotechnology Information Gene Expression Omnibus (NCBI GEO): GSE12493 ) consisting of 2 independent SSc fibroblast cell lines, 1 healthy control fibroblast cell line (isolated in parallel), and 1 normal human dermal (NHD) fibroblast cell line obtained from American Type Culture Collection (ATCC), treated with 50 pM TGF- β 1 [ ] ( ).

Techniques: Expressing, Inhibition, Western Blot, Isolation, Control, Single Cell, RNA Sequencing, Marker

Journal: Annals of the rheumatic diseases

Article Title: RUNX1 is expressed in a subpopulation of dermal fibroblasts and is associated with disease severity of systemic sclerosis

doi: 10.1016/j.ard.2025.10.033

Figure Lengend Snippet: RUNX1 contributes to fibroblast activation, proliferation and contraction. (A) RUNX1 western blot of CRISPR-generated RUNX1 KO and wild-type (WT) fibroblasts under the TGF- β 1 stimulation vs control. RUNX1 isoforms of a, b, and c were marked in the blot by arrows. (B) α -SMA and RUNX1 IF staining of KO and WT fibroblasts under the TGF- β 1 stimulation vs control. (C) α -SMA western blot of KO and WT fibroblasts under the TGF- β 1 stimulation vs control. (D) ACTA2 mRNA expression of KO and WT fibroblasts under the TGF- β 1 induction vs control. (E) Fold change expression of FN1, COL1A1, LUM , and SFRP4 in TGF- β 1-induced SSc fibroblasts treated with Ro5–3335 compared to control (3 lines of SSc fibroblasts, 2 replicates each). (F) Proliferation curve of normal human dermal (NHD) fibroblasts in the presence and absence of Ro5–3335. (G,H) The 3D collagen contraction assays, fixed (G) and floating (H) models, of NHD fibroblasts treated with Ro5–3335 (4 replicates for each condition). SIS3 (SMAD3 inhibitor) was used as positive control that significantly eliminates the contraction ability of fibroblasts. Negative control is collagen matrix with no fibroblasts. The overhead pictures represent 1 replicate for each condition. (I) 3D self-assembled (SA) tissue constructs from the healthy- and SSc-isolated fibroblast lines with donors’ clinical characteristics. H&E staining of representative untreated and Ro5–3335-treated tissues. (J) Tissue area fold change of each cell line over the control for healthy and SSc SA tissues. Data from 3 healthy and 4 SSc lines, 3 replicates per line, repeated in 2 independent sets. (K) Change in area of an SSc-isolated SA tissue when treated for 1, 2, or 3 weeks with Ro5–3335 compared to control (Student’s t test P value: **.001-.01, ****<.0001 in GraphPad Prism v9). α -SMA, alpha smooth muscle actin; H&E, haematoxylin and eosin; KO, knockout; RUNX1, runt-related transcription factor 1; SSc, systemic sclerosis; TGF- β , transforming growth factor- β ;Clustered Regularly Interspaced Palindromic Repeats (CRISPR),Smad Family Member 3 (SMAD3),Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH), Not Applicable (N/A), Quantitative Polymerase Chain Reaction (QPCR), Quantitative Polymerase Chain Reaction, Immunofluorescenc (IF).

Article Snippet: We then analysed a previously generated DNA microarray dataset (National Center for Biotechnology Information Gene Expression Omnibus (NCBI GEO): GSE12493 ) consisting of 2 independent SSc fibroblast cell lines, 1 healthy control fibroblast cell line (isolated in parallel), and 1 normal human dermal (NHD) fibroblast cell line obtained from American Type Culture Collection (ATCC), treated with 50 pM TGF- β 1 [ ] ( ).

Techniques: Activation Assay, Western Blot, CRISPR, Generated, Control, Staining, Expressing, Positive Control, Negative Control, Construct, Isolation, Knock-Out, Real-time Polymerase Chain Reaction